The goal of cell division (mitosis) is to partition genetic information, in the form of chromosomes, equally into the two daughter cells. The overarching goal of our research is to reveal the mechanisms that allow the spindle to assemble rapidly and with minimal number of errors. Our previous work demonstrates that every major step in spindle assembly can be reached via several alternative routes. This multiplicity of alternative mechanisms prompts the hypothesis that a proper balance in the contributions from individual mechanisms must be maintained to ensure error-free chromosome segregation. The goal of the parent 1 R35 GM130298-01 (Efficiency and fidelity in mitotic spindle assembly) is to test this hypothesis by quantitatively characterizing spindle assembly in normal vs. chromosomally instable (CIN) cells that frequently mis-segregate their chromosomes. Towards this goal, we identify and characterize molecular mechanisms that govern attachment of chromosomes to microtubules of the mitotic `spindle' via sophisticated imaging such as laser microsurgery, precise tracking of chromosome movements, and correlative electron-microscopy analyses conducted on the kinetochores whose behavior was followed in live cells up to the moment of fixation. These approaches rely on a laser microirradiation workstation, designed, assembled, and maintained in the PI's laboratory with support from the NIH grants and funds contributed by the Wadsworth Center. This application seeks to obtain funds for upgrading the workstation to enable laser operations in cells with various organelles labeled in different colors. Multi-color capabilities will vastly accelerate our scientific progress towards the goals of the parent grant and will also facilitate several collaborative studies supported by NIH GMS grants.